Use of the Pharmacological Inhibitor BX795 to Study theRegulation and Physiological Roles of TBK1 and I�B Kinase �A DISTINCT UPSTREAM KINASE MEDIATES SER-172 PHOSPHORYLATION AND ACTIVATION*□S

Received for publication, January 5, 2009, and in revised form, March 3, 2009 Published, JBC Papers in Press, March 25, 2009, DOI 10.1074/jbc.M109.000414

Kristopher Clark1, Lorna Plater, Mark Peggie, and Philip Cohen2

From the MRC Protein Phosphorylation Unit, College of Life Sciences, Sir James Black Centre, University of Dundee,Dundee DD1 5EH, Scotland, United Kingdom

TANK-binding kinase 1 (TBK1) and I�B kinase � (IKK�) reg-ulate the production of Type 1 interferons during bacterial andviral infection, but the lack of useful pharmacological inhibitorshas hampered progress in identifying additional physiologicalroles of these protein kinases and how they are regulated. Herewe demonstrate that BX795, a potent and relatively specificinhibitor of TBK1 and IKK�, blocked the phosphorylation,nuclear translocation, and transcriptional activity of interferonregulatory factor 3 and, hence, the production of interferon-� inmacrophages stimulated with poly(I:C) or lipopolysaccharide(LPS). In contrast, BX795 had no effect on the canonical NF�Bsignaling pathway. Although BX795 blocked the autophospho-rylation of overexpressedTBK1and IKK� at Ser-172 and, hence,the autoactivation of these protein kinases, it did not inhibit thephosphorylation of endogenous TBK1 and IKK� at Ser-172 inresponse to LPS, poly(I:C), interleukin-1� (IL-1�), or tumornecrosis factor � and actually enhanced the LPS, poly(I:C), andIL-1�-stimulated phosphorylation of this residue. These resultsdemonstrate that the phosphorylation of Ser-172 and the acti-vation of TBK1 and IKK� are catalyzed by a distinct proteinkinase(s) in vivo and that TBK1 and IKK� control a feedbackloop that limits their activation by LPS, poly(I:C) and IL-1� (butnot tumor necrosis factor �) to prevent the hyperactivation ofthese enzymes.

Invading bacteria and viruses are sensed by the host patternrecognition receptors, which bind components of these orga-nisms, called pathogen-associated molecular patterns. Thebinding of pathogen-associated molecular patterns to patternrecognition receptors activates signaling cascades that culmi-nate in the production of proinflammatory cytokines, chemo-kines, and interferons, which are released from immune cellsinto the circulation, where theymount responses to combat theinvading pathogen (1). The interaction between pathogen-as-sociated molecular patterns and pattern recognition receptorsleads invariably to the activation of the mitogen-activated pro-

tein (MAP)3 kinases, termed p38MAPkinases and c-JunN-ter-minal kinases 1 and 2 (JNK1/2) and the I�B kinase (IKK) com-plex. The latter contains the protein kinases IKK� and IKK�,which switch on the transcription factor NF�B and, hence,NF�B-dependent gene transcription, by phosphorylating I�B�and other I�B isoforms (2). IKK� also activates the proteinkinase Tpl2 by phosphorylating its p105 regulatory subunit,leading to the activation of two other MAP kinases, termedextracellular signal-regulated kinase 1 (ERK1) and ERK2 (3, 4).Together, the MAP kinases and NF�B regulate the productionof many proinflammatory cytokines and chemokines.A subset of pattern recognition receptors, namely Toll-like

receptors 3 and 4 (TLR3, TLR4) and the cytosolic receptorsRIG-I (retinoic acid-inducible gene I) and MDA-5 (melanomadifferentiation-associated gene 5), also activate a distinct sig-naling pathway requiring the IKK-related kinases, IKK� andTANK-binding kinase 1 (TBK1) (5, 6). Early studies, largelybased on overexpression experiments, suggested that a majorrole of TBK1 and IKK� was to activate NF�B and NF�B-de-pendent gene transcription, and for this reason, TBK1 has alsobeen called NF�B-activating kinase (7–9). However, later stud-ies using cells frommice that do not express TBK1 and/or IKK�failed to support this conclusion (10, 11). Instead, they indi-cated that these protein kinases play an essential role in regu-lating the production of type I interferons (IFNs) by phospho-rylating the transcription factor, termed interferon regulatoryfactor 3 (IRF3) (10, 11). Under basal conditions IRF3 is cytoso-lic, but after the TBK1/IKK�-mediated phosphorylation of its Cterminus, IRF3 dimerizes and translocates to the nucleus,where it activates a gene transcription program leading to theproduction of IFN-� (12, 13). The production of IFN-� mayadditionally require the TBK1/IKK�-catalyzed phosphoryla-tion of other proteins, such as the Dead-box RNA-helicaseDDX3 (14, 15) andMITA (16). IKK� has also been implicated inthe phosphorylation of the STAT1 transcription factor at Ser-708 in a pathway that protects cells against infection by influ-enza A virus (17).

* The work was supported by the United Kingdom Medical Research Council,AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Merck-Serono, andPfizer.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. S1.

1 Recipient of a long term fellowship from EMBO.2 A Royal Society Research Professor. To whom correspondence should be

addressed. Tel.: 44-1382-384238; Fax: 44-1382-223778; E-mail: p.cohen@dundee.ac.uk.

3 The abbreviations used are: MAP, mitogen-activated protein; MAPK, MAPkinase; MKK, MAPK kinase; MAP3K, MAPK kinase kinase; ASK1, apoptosis-stimulating kinase-1; ERK, extracellular signal-regulated kinase; IFN, inter-feron; IKK, I�B kinase; IRF3, interferon regulatory factor 3; JNK1/2, c-JunN-terminal kinase; LPS, lipopolysaccharide; MLK, mixed lineage kinase;PDK1, 3-phosphoinositide-dependent protein kinase 1; TAK1, transform-ing growth factor �-activated kinase 1; TBK1, TANK-binding kinase 1; TLR,Toll-like receptor; TNF, tumor necrosis factor; IL, interleukin; MEF, mouseembryonic fibroblast; GST, glutathione S-transferase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 21, pp. 14136 –14146, May 22, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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However, mouse knock-out studies are not always definitivebecause the complete loss of a protein kinase(s) may be com-pensated for by other protein kinases, whereas the prolongedabsence of a protein kinase may result in long term changes ingene transcription programs so that the effects observedmay beindirect. The embryonic lethality of the TBK1 knock-outmouse also limits its use in understanding the physiologicalroles of this protein kinase. Moreover, papers continue to bepublished proposing roles for TBK1 and IKK� in phosphoryl-ating defined sites on the RelA and c-Rel components of theNF�B transcription complex that are thought to control theexpression of a subset of NF�B-dependent genes (18–20).Finally, there is considerable evidence that TBK1 and IKK� playadditional roles in cells. For instance, TBK1 is activated byTNF,andTBK1 knock-outmice die just before birth because the fetalhepatocytes undergo TNF�-induced apoptosis (21). Theseobservations imply that TBK1 plays a key role in preventingapoptosis in the fetal hepatocytes of wild type mice. TBK1 isalso reported to be activated by hypoxia and to control theproduction of angiogenic factors, such as vasoendothelialgrowth factor (22), whereas the overexpression of IKK� inbreast cancer lines is reported to contribute a survival signal tothe transformed cells (23). The direct substrates of TBK1 andIKK� or molecular pathways underlying any of these responsesand the possible roles of these protein kinases in the pathogen-esis of human cancer are unknown.The identification of the physiological substrates and biolog-

ical roles of protein kinases has been greatly aided by the use ofrelatively specific, small cell-permeable inhibitors of theseenzymes. These compounds can be used simply and rapidly andprovide a complementary approach to the use of mouse knock-outs or RNA interference technology, avoiding the potentialdrawbacks in ablating the expression of a protein kinase thatwere mentioned above. The compound BX795 was originallydeveloped as a small molecule inhibitor of 3-phosphoinositide-dependent protein kinase 1 (PDK1) (24), but we recently foundthat it also inhibited TBK1 and IKK� at low nanomolar concen-trations in vitro (25). Moreover, BX795 only inhibited a fewother protein kinases significantly out of 70 tested and, impor-tantly, did not inhibit IKK� (25). These findings suggested thatBX795might be the first pharmacological inhibitor suitable forstudying the regulation and roles of TBK1 and IKK� in cells. Inthis paper we demonstrate the utility of this compound andexploit it to show that, unexpectedly, the activation of TBK1and IKK� is not an autophosphorylation event but is mediatedby a distinct “upstream” protein kinase.

EXPERIMENTAL PROCEDURES

Materials—BX795 was synthesized as described (25), dis-solved in DMSO, and stored as a 10 mM solution at �20 °C.Poly(I:C) and LPS were from InvivoGen, and mouse IL-1� andTNF� were from Sigma.DNAConstructs—TBK1 (NCBI NP_037386.1) was amplified

from IMAGE EST 5492519 (Geneservice) using KODHot StartDNAPolymerase (Novagen). The PCR product was cloned intopSC-b (Stratagene) and sequenced to completion. The insertwas excised using BamHI and NotI and inserted into pCMV-FLAG-1 or pEBG6P to generate FLAG-TBK1 and GST-TBK1,

respectively. IKK� (NCBI NP_054721.1) was cloned in a similarmanner using IMAGE EST 3062062. Pointmutations were cre-ated using the QuikChange mutagenesis kit (Stratagene) butusing KOD Hot Start DNA Polymerase. IRF3 (GenBankTMCAA91227.1) was amplified by PCR using IMAGE EST5494536, ligated with pSC-b, and sequenced. The insert wassubcloned into NotI sites of pGEX6P-2. PRDII (NF�B) andPRDIII-I (IRF3) elements from the IFN-� promoter cloned intothe pLuc-MCS vector were a kind gift from Katherine Fitzger-ald (University of Massachusetts). pTK-RL was obtained fromStratagene.Cell Culture—HEK293 cells stably expressing TLR3-FLAG

(termed HEK293-TLR3 cells) were provided by KatherineFitzgerald (University of Massachusetts), immortalized mouseembryonic fibroblasts (MEFs) were from wild type mice, andmice expressing a truncated, inactive form of TAK1 (26) wereprovided by Professor Shizuo Akira (Osaka University, Japan).HEK293-TLR3, RAW264.7 cells (hereafter termed RAW cells)and MEFS were maintained in Dulbecco’s modified Eagle’smedium supplemented with 2 mM glutamine, 10% fetal calfserum, and the antibiotics penicillin and streptomycin. Bone-marrow derived macrophages were generated from mice asdescribed (27). Cells were transfected using Lipofectamine2000 (Invitrogen) according to themanufacturer’s instructions.Antibodies—Antibodies were raised in sheep against the

human TBK1 protein expressed in insect Sf21 cells (SheepS041C, bleed 2) (25) and the C-terminal peptide of mouse IKK�(NRLIERLHRVPSAPDV) (Sheep S277C, bleed 2) and used forimmunoprecipitation. The phosphopeptide CEKFVS*VYGTE(where S* indicates phosphoserine) corresponding to thesequence surrounding Ser-172 of TBK1 and IKK� was used togenerate an antibody that immunoprecipitated the phosphoryl-ated forms of these protein kinases (Sheep S051C, bleed 2). Thephosphopeptide was coupled separately to keyhole limpethemocyanin and bovine serum albumin, then mixed andinjected into sheep at Diagnostics Scotland (Edinburgh, UK).The antisera were purified by affinity chromatography onimmobilized TBK1 or the peptide antigen, respectively, in theDivision of Signal Transduction Therapy (University ofDundee. The phospho-specific antibody was incubated withthe unphosphorylated form of the peptide immunogen (10 �gof peptide per �g of antibody) before use to neutralize any anti-bodies recognizing the unphosphorylated forms of TBK1 andIKK�. The following antibodies were used for immunoblotting:anti-glyceraldehyde-3-phosphate dehydrogenase (ResearchDiagnostics Inc.), anti-GST (Division of Signal TransductionTherapy, University of Dundee), horseradish peroxidase-con-jugated secondary antibodies (Pierce), anti-IRF3, anti-TAK1(Santa Cruz), anti-FLAG, anti-IKK� (Sigma), anti-TBK1, anti-TANK and anti-I�B� (Cell Signaling Technology). Antibodiesrecognizing phosphorylated Ser-933 (Ser(P)-933) of p105(NF�B1), Ser(P)-32/Ser(P)-36 of I�B�, Ser(P)-468 of RelA,Ser(P)-536 of RelA, Ser(P)-396 of IRF3, the Thr(P)-Glu-Tyr(P)sequence of ERK1 and ERK2, the Thr(P)-Gly-Tyr(P) sequenceof p38MAP kinases and a pan-PDK1 phosphorylation site anti-body which recognizes the phosphorylated activation loop(Thr(P)-229) of S6 kinase 1 (28) were also from Cell SignalingTechnology. The antibody recognizing the Thr(P)-Pro-Tyr(P)

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sequence of JNK1/2 was from BIOSOURCE, whereas that rec-ognizing Ser(P)-172 of TBK1 was from BD Biosciences.Immunoprecipitation and Immunoblotting—Pharmacologi-

cal inhibitors dissolved in DMSO or an equivalent volume ofDMSO for control incubations were added to the culturemedium of cells grown as monolayers. After 1 h at 37 °C, thecells were stimulated with LPS, poly(I:C), IL-1�, or TNF� asdescribed in all the figure legends. Thereafter, the cells wererinsed in ice-cold phosphate-buffered saline and extracted inlysis buffer (50mMTris/HCl, pH 7.4, 1mMEDTA, 1mMEGTA,50 mM NaF, 5 mM sodium pyrophosphate, 10 mM sodium�-glycerol 1-phosphate, 1 mM dithiothreitol, 1 mM sodiumorthovanadate, 0.27 M sucrose, 1% (v/v) Triton X-100, 1 mg/mlaprotinin, 1 mg/ml leupeptin, 1 mM phenylmethylsulfonyl flu-oride). Cell extracts were clarified by centrifugation at 14,000�g for 10 min at 4 °C, and protein concentration was determinedusing the Bradford assay. Proteinswere immunoprecipitated byincubating 1 mg of cell extract protein with 10 �g of antibodyfor 90 min at 4 °C followed by the addition of Protein G-Sepha-rose. After mixing for 15 min at 4 °C and brief centrifugation,the immunocomplexes were washed 3 times in lysis buffer,denatured in SDS, and subjected to SDS-PAGE. To detect pro-teins in cell lysates, 40 �g of protein extract was separated bySDS-PAGE. After transfer to polyvinylidene difluoride mem-branes, proteins were detected by immunoblotting and visual-ized by treating the blots with ECL (Amersham Biosciences)followed by autoradiography.Protein Kinase Assays—IRF3 was expressed in Escherichia

coli as a GST fusion protein and purified by affinity chromatog-raphy on glutathione-Sepharose. Endogenous TBK1 wasimmunoprecipitated from 1 mg of cell lysate protein using thesheep anti-TBK1 (sheep S041C, bleed 2) antibody and overex-pressed FLAG-TBK1 with an anti-FLAG M2-agarose (Sigma).Immunocomplexes were washed 3 times in lysis buffer andtwice in 50mMTris-HCl, pH7.5, 0.1% (v/v) 2-mercaptoethanol,0.1 mM EGTA, 10 mM magnesium acetate and then resus-pended in the same buffer containing 2 �M GST-IRF3 (endog-enous TBK1) or the peptide KKKKERLLDDRHDSGLDSMK-DEE (0.3 mM), corresponding to the sequence surroundingSer-32 and Ser-36 of I�B�, plus four lysine residues at the Nterminus to facilitate binding to phosphocellulose paper(termed I�B� peptide substrate). Assays were initiated by add-ing [�-32P]ATP (1000 cpm/pmol) to a final concentration of 0.1mM. When GST-IRF3 was used as the substrate, the reactionswere terminated after 30 min at 30 °C by the addition of SDScontaining 40mMEDTA, pH7.0, heated for 5min at 100 °C andseparated by SDS-PAGE, and phosphorylated proteins weredetected by autoradiography. Quantification was performed byphosphorimaging analysis. For assays with the I�B� substratepeptide, reactions were terminated after 10 min at 30 °C byspotting an aliquot of the reaction on to a 2 � 2-cm2 piece ofphosphocellulose P81 paper (Whatman) followed by immer-sion in 75mMphosphoric acid. After washing six times in phos-phoric acid and once in acetone, the papers were dried andcounted. One unit of TBK1 activity was defined as that amountof enzyme catalyzing the incorporation of 1 nmol of phosphateinto substrate in 1 min.

Luciferase Assays—Cells were co-transfected with PRD(II)2or PRD(III-I)3-pLuc-MCS and pTK-RL plasmid DNA. 24 hlater cells were stimulated and extracted in Passive Lysis Buffer(Promega). Luciferase activity was measured with a dual-lucif-erase assay system (Promega) according to the manufacturer’sinstructions.Measurement of IFN-� Production—Cells were treated for

1 h with or without inhibitors then stimulated for 6 h with 100ng/ml LPS or 10 �g/ml poly(I:C). The cell culture medium wasremoved and clarified by centrifugation for 10 min at 14,000 �g, and the concentration of mouse IFN-� was measured usingan enzyme-linked immunosorbent assay kit (R&D Systems)according to manufacturer’s protocol.Microscopy—Cells seeded on glass coverslips were serum-

starved overnight before stimulation with poly(I:C). Thecells were fixed, permeabilized, and stained as described (29)using anti-IRF3 (1:100; Santa Cruz) followed by incubationwith Alexa546-conjugated anti-rabbit IgG (1:500; MolecularProbes). The cells were then visualized using a Zeiss-LSM 510-meta microscope fitted with an alpha Plan-Fluar 100x/1.45 oilobjective.Statistical Analysis—Quantitative data are presented as the

mean � S.E. Statistical significance of differences betweenexperimental groups was assessed with Student’s t test. Differ-ences in means were considered significant if p � 0.05.

RESULTS

Specificity of BX795 in Vitro—We reported previously thatsix protein kinases were inhibited by �90% in vitro in the pres-ence of 0.1 �M BX795, namely TBK1, IKK�, PDK1, Aurora B,ERK8, and MARK3, but 60 other protein kinases tested wereunaffected or only slightly inhibited at this concentration (25).The IC50 values for these six protein kinases are shown in Table1. MARK3 is a member of the subgroup of protein kinases thatinclude the AMP-activated protein kinase. In the present studywe found that the other MARK isoforms (MARK1, MARK2,and MARK4) and another AMP-activated protein kinase-re-lated kinase (NUAK1) were inhibited with similar potency toMARK3 (Table 1). We also studied the effect of BX795 on anumber of additional kinases not examined previously. These

TABLE 1I50 values for the inhibition of selected protein kinases by BX795Data are presented as the average of duplicate determinations. VEGFR, vasoendo-thelial growth factor receptor.

Kinase IC50 ATP�M �M

TBK1 0.006 � 0.001 100IKK� 0.041 � 0.001 100PDK1 0.111 � 0.013 100Aurora B 0.031 � 0.002 100ERK8 0.140 � 0.027 100MARK3 0.081 � 0.008 100MARK1 0.055 � 0.004 100MARK2 0.053 � 0.005 100MARK4 0.019 � 0.001 100NUAK1 0.005 � 0.001 100VEGFR 0.157 � 0.011 100MLK1 0.050 � 0.003 100MLK2 0.046 � 0.011 100MLK3 0.042 � 0.005 100TAK1 0.70 � 0.14 100ASK1 0.620 � 0.004 100

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experiments showed that BX795 did not inhibit the followingprotein-tyrosine kinases at 1 �M: ephrin receptors A2 and B3,Syk, Bruton’s tyrosine kinase, and fibroblast growth factorreceptor 1. However, the vascular endothelial growth factorreceptor was inhibited, albeit much less potently than TBK1(Table 1). We reported previously that BX795 did not inhibit

IKK� (25), and in the present studywe found that the IKK� isoform isalso unaffected at 1 �M in vitro(results not shown). For reasons dis-cussed later, we also examined theeffect of BX795 on a number ofMAP kinase kinases (MKKs) andMAP kinase kinase kinases (termedMAP3Ks). At 1 �M, BX795 did notinhibit MKK1, MKK3, MKK4,MKK6, and MKK7, or the MAP3KsTpl2 and c-Raf (results not shown)but did potently inhibit the mixedlineage kinases, termed MLK1(MAP3K9),MLK2 (MAP3K10), andMLK3 (MAP3K11)) (Table 1).MAP3K7, also called transforminggrowth factor �-activated kinase-1(TAK1), and MAP3K5, also calledapoptosis-stimulating kinase-1 (ASK1),were inhibited �100-fold lesspotently than TBK1 (Table 1). Theability of BX795 to inhibit theTBK1-catalyzed phosphorylation ofIRF3 at Ser-396 declined as the ATPconcentration in the assay wasincreased (supplemental Fig. S1),indicating that BX795 is an ATPcompetitive inhibitor of TBK1 as isthe case for PDK1 (24).BX795 Blocks TBK1- and IKK�-

mediated Activation of IRF3 andProduction of IFN-�—To examinewhether BX795 inhibited TBK1 andIKK� when added to mammaliancells in culture, we used HEK293cells that stably overexpress TLR3(termed HEK293-TLR3 cells) forinitial studies. IRF3 migrated as adoublet in unstimulated cells. Stim-ulation with poly(I:C), a syntheticTLR3 agonist, led to the appearanceof a more slowly migrating specieswhose level became maximal after2 h (Fig. 1A). The appearance of thisspecies, which has been shown byothers to result from phosphoryla-tion (30), was prevented by priorincubation with BX795 (Fig. 1A).We also observed that IRF3 accu-mulated in the nucleus on a similartime scale after poly(I:C) treatment,

which was blocked by BX795 (Fig. 1B). The next step in thispathway is IRF3-stimulated gene transcription. As expectedfrom the results presented above, BX795 inhibited IRF3-de-pendent gene transcription (Fig. 1C). Finally, BX795 blockedthe secretion of IFN-� from macrophages whether stimulatedby LPS, a TLR4 agonist (Fig. 1, D and E), or poly(I:C) (Fig. 1E).

FIGURE 1. BX795 blocks the phosphorylation, nuclear translocation, and transcriptional activity of IRF3and production of interferon � in response to TLR3 and TLR4 agonists. A, HEK293-TLR3 cells were incu-bated for 60 min without (�) or with (�) 1 �M BX795 and subsequently stimulated with 50 �g/ml poly(I:C) forthe times indicated. Cell extract protein (40 �g) was subjected to SDS-PAGE and immunoblotted for IRF3.B, HEK293-TLR3 cells were stimulated for 2 h with poly(I:C) as in A. The cells were fixed, stained for IRF3, andvisualized by confocal microscopy. C, HEK293-TLR3 cells were co-transfected with DNA encoding an IRF3luciferase reporter construct and pTK-Renilla luciferase plasmid DNA. 24 h post-transfection cells were incu-bated for 1 h with varying concentrations of BX795 before stimulation for 6 h with 50 �g/ml poly(I:C). Luciferaseactivity was measured and normalized to Renilla luciferase activity (mean � S.E., n � 4). D, RAW264.7 cells wereincubated for 1 h at various concentrations of BX795 and then stimulated for 6 h with 100 ng/ml LPS. Theconcentration of IFN-� released into the culture medium was measured by enzyme-linked immunosorbentassay. (mean � S.E., n � 4). E, the experiment was carried out as in D except that cells were incubated without(white bars) or with (black bars) 1 �M BX795 and stimulated with either 100 ng/ml LPS or 10 �g/ml of poly(I:C).(mean � S.E., n � 3; *, p � 0.01). F, the effects of BX795 on TLR signaling do not result from inhibition of PDK1.RAW264.7 macrophages were treated for 30 min without (control) or with 1 �M BX795 then stimulated for 45min without (�) or with (�) 100 ng/ml LPS. Cell extracts (40 �g protein) were immunoblotted with antibodiesrecognizing IRF3, IRF3 phosphorylated at Ser-396, and S6 kinase 1 (S6K1) phosphorylated at Thr-229.

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BX795 was originally developed as an inhibitor the proteinkinase PDK1, but BX795 had no effect on the LPS-stimulatedphosphorylation of p70 ribosomal S6 kinase 1 at Thr-229, thesite that is targeted by PDK1 (31), under conditions where itcompletely blocked the LPS-stimulated phosphorylation ofIRF3 at Ser-396 (Fig. 1F). This experiment demonstrated thatTBK1/IKK� was inhibited much more potently than PDK1 byBX795 in cells and excluded the possibility that BX795 sup-pressed the activation of IRF3 and production of IFN-� byinhibiting PDK1.BX795 Does Not Affect Activation of the IKK�/� Complex or

NF�B-dependent Gene Transcription by LPS, poly(I:C), IL-1�,or TNF�—The question of whether TBK1 and IKK� play arole in regulating NF�B-dependent gene transcription is anissue that is still not fully resolved (see the Introduction). We,

therefore, studied the effect ofBX795 on the activation of NF�Band NF�B-dependent transcrip-tional activity. Under conditionswhere BX795 completely blockedthe LPS (Fig. 2A)- or poly(I:C)-stim-ulated (Fig. 2B) phosphorylation ofIRF3 at Ser-396, this compound didnot affect the phosphorylation atSer-32 and Ser-36 or degradation ofthe I�B� inhibitory component ofthe NF�B transcription complexor the phosphorylation at Ser-933of the p105 (NF�B1) regulatorysubunit of the protein kinase Tpl2(Fig. 2, A and B), which are estab-lished physiological substrates ofthe IKK�/� complex. These resultsdemonstrated that BX795 did notinhibit any of the steps involved inthe LPS- or poly(I:C)-stimulatedactivation of the IKK�/� complexor the ability of IKK�/� to phospho-rylate downstream substrates. Nordid BX795 inhibit the LPS- orpoly(I:C)-stimulated phosphoryla-tion of the RelA component of theNF�B transcription factor atSer-468 and Ser-536 (Fig. 2, A andB), amino acid residues reportedto become phosphorylated whenTBK1 and/or IKK� were overex-pressed in cells (18, 19). BX795 didnot inhibit NF�B-dependent genetranscription in response topoly(I:C) in HEK293-TLR3 cells(Fig. 2C), which is consistent withthe failure of BX795 to suppress theactivation or activity of the IKK�/�complex or the phosphorylation ofRelA. BX795 also had no effect onthe activation of the canonicalNF�B pathway by IL-1� and TNF�

(Fig. 2, D and E), although TBK1 and IKK� are activated asdiscussed later.Activation of TBK1 and IKK�Correlates with the Phosphoryl-

ation of Ser-172—The molecular mechanisms that lead to theactivation of TBK1 and IKK� are still rather poorly defined,although the activation of these protein kinases is known torequire their phosphorylation at Ser-172 within the activationloop (9, 15, 32). In the experiments described below we initiallymonitored the increase in the catalytic activity of TBK1 andIKK� by direct enzyme assay as well as by the phosphorylationof Ser-172 (Fig. 3). LPS stimulation of RAW macrophagesincreased the catalytic activity of TBK1, which was maximalafter 30 min, persisted until 60 min, and correlated with thephosphorylation of Ser-172 (Fig. 3A). Similarly, poly(I:C) stim-ulation ofHEK293-TLR3 cells led to an increase in TBK1 activ-

FIGURE 2. BX795 selectively blocks IRF3 but not NF�B signaling. A, RAW264.7 cells were incubated without(�) or with (�) 1 �M BX795 and then either left unstimulated (�) or stimulated (�) for 30 min with 100 ng/mlLPS. Cell extracts (40 �g protein) were then immunoblotted with the antibodies used in Fig. 1 and withantibodies that recognize TBK1, RelA phosphorylated at Ser-468 or Ser-536, I�B� phosphorylated at Ser-32 andSer-36, or p105 phosphorylated at Ser-933. B, same as A except that the RAW264.7 cells were stimulated with 10�g/ml poly(I:C) for 60 min, and immunoblotting for I�B� was carried out using an antibody recognizing allforms of I�B� instead of the antibody recognizing phosphorylated I�B�. C, HEK293-TLR3 cells were co-trans-fected with DNA encoding an IRF3 or a NF�B luciferase reporter construct and pTK-Renilla luciferase plasmidDNA. 24 h post-transfection cells were treated without (white bars) or with (black bars) 1 �M BX795 and thenstimulated for 6 h with 50 �g/ml poly(I:C). Luciferase activity was measured and normalized to Renilla luciferaseactivity (mean � S.E., n � 4; *, p � 0.001). D and E, MEFs were serum-starved overnight and incubated without(�) or with (�) 1 �M BX795 for 60 min before stimulation with 10 ng/ml IL-1� (D) or TNF-� (E). Cell extracts wereimmunoblotted as described in A and B.

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ity after 60 min that was maximal after 120 min and againcorrelated with the phosphorylation of Ser-172 (Fig. 3B).The sequence surrounding Ser-172 is conserved between

TBK1 and IKK�. The phospho-specific antibody raised againstthe sequence surrounding Ser-172 should, therefore, re-cognize the phosphorylated forms of both protein kinases. Wefound that the anti-phospho-Ser-172 antibody that we generatedimmunoprecipitated phosphorylated TBK1 and IKK�, allowing

the phosphorylation of these protein kinases to be monitored bysubsequent immunoblotting with antibodies that recognize allformsofTBK1and IKK�. Thismethodalso enabledchanges in thephosphorylation of TBK1 and IKK� to be studied simultaneously,as TBK1migratedmore slowly than IKK� during SDS-PAGE (Fig.3C). These studies revealed that TBK1 and IKK�were activated inparallel in response to LPS (Fig. 3C).Transfected Catalytic Subunits of TBK1 and IKK� Are Acti-

vated by Autophosphorylation—It was reported that the trans-fected catalytic subunit ofTBK1becamephosphorylated at Ser-172 but catalytically inactive mutants did not, which has led tothe assumption that phosphorylation of Ser-172 is an auto-phosphorylation event (9, 15). These observations with trans-fected TBK1 were confirmed in the present study (Fig. 4A),where we also found that BX795 prevented the phosphoryla-tion of Ser-172 (Fig. 4B) and the activation of transfected TBK1(Fig. 4C). Additionally, we found that the phosphorylation ofTBK1 at Ser-172 is at least partly an intermolecular autophos-phorylation event, because wild type TBK1 associated with andphosphorylated the catalytically inactivemutant TBK1-(K38A)in co-transfection experiments (Fig. 4D). Moreover, TBK1-(K38A) also interacted with and could be phosphorylated bywild type IKK� (Fig. 4D).

Although the transfected wild type and catalytically inactivemutants of TBK1 were expressed at similar levels, we noticedthat wild type IKK� accumulated to levels that were far greaterthan the catalytically inactive mutants (Fig. 4E). Moreover,unlike TBK1 (Fig. 4B), incubation of the cells expressing wildtype Flag-IKK� with BX795 led to a rapid decrease in the pro-tein to the levels observed when a catalytically inactive mutantwas expressed (Fig. 4F). Furthermore, incubation with BX795did not decrease the expression of the catalytically inactivemutant of IKK� further (results not shown). Taken together,these experiments demonstrate that the phosphorylationand/or the catalytic activity of IKK� is required for the stabilityof the transfected protein kinase.The level of expression of the endogenous IKK� is

increased by prolonged exposure to inflammatory stimuli,and for this reason, it is also called IKK-inducible (IKKi) (9).We found that the LPS-induced increase in the expression ofendogenous IKK� was not decreased by BX795 (Fig. 5A),demonstrating that it is not dependent on IKK� catalyticactivity. Moreover, in contrast to the overexpressed catalyticsubunits, immunoprecipitation of endogenous IKK� did notcoimmunoprecipitate TBK1 or vice versa (Fig. 5B). Immu-noprecipitation of IKK� or TBK1 also led to the coimmuno-precipitation of TANK (Fig. 5B), as expected from earlierreports (8). Taken together, these results indicate that theendogenous TBK1 and IKK� are present in RAW macro-phages as separate complexes.Phosphorylation of Endogenous TBK1 and IKK� at Ser-172

Is Mediated by a Distinct Protein Kinase(s)—We next stud-ied the activation (Fig. 6A) and Ser-172 phosphorylation(Fig. 6B) of the endogenous TBK1 and IKK� in LPS-stimu-lated RAWmacrophages. Surprisingly, and in complete con-trast to the transfection experiments described above,BX795 not only failed to reduce the phosphorylation of theendogenous protein kinases at Ser-172 but actually

FIGURE 3. Phosphorylation of TBK1 and IKK� at Ser-172 correlates withcatalytic activity. A, RAW264.7 macrophages were stimulated with 100ng/ml LPS for the times indicated. Catalytic activity was measured by immu-noprecipitating TBK1 and incubating the immunocomplexes with GST-IRF3in presence of Mg[�-32P]ATP. Proteins were resolved by SDS-PAGE andstained with Coomassie Blue, and phosphorylated IRF3 detected by autora-diography (top panel). Phosphorylation of Ser-172 on TBK1 was monitored byimmunoprecipitating the phosphorylated TBK1 with the anti-IKK� Ser(P)-172antibody and immunoblotting with an antibody recognizing all forms ofTBK1 (middle panel). A further aliquot of cell extract was subjected to SDS-PAGE and immunoblotted with the same TBK1 antibody. B, same as A, exceptthat HEK293-TLR3 cells were stimulated for the times indicated with 50 �g/mlpoly(I:C). C, RAW264.7 macrophages were left unstimulated (�) or stimulated(�) for 45 min with 100 ng/ml LPS. The Ser-172-phosphorylated forms of TBK1and IKK� were immunoprecipitated from cell extracts using the anti-Ser(P)-172 IKK� antibody, and the presence of TBK1 and/or IKK� was revealed byimmunoblotting using antibodies recognizing all forms of TBK1 (top panel) orIKK� (second panel from top) or a mixture of the two antibodies (third panelfrom the top). In the bottom panel, 40 �g of extract protein was immuno-blotted without immunoprecipitation using a mixture of the antibodies rec-ognizing all forms of TBK1 and IKK�.

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enhanced phosphorylation (Fig. 6,B and C) and activation (Fig. 6A)by about 2-fold. Similar resultswere obtained in primary bonemarrow-derived macrophagesstimulated with LPS (Fig. 6D) orpoly(I:C) (Fig. 6E).The different effects of BX795

on the phosphorylation of Ser-172of endogenous and transfectedTBK1/IKK� raised the question ofwhether the endogenous kinaseswere insensitive to BX795. Al-though this seemed unlikely, asBX795 suppresses the phosphoryla-tion of the TBK1/IKK� substrateIRF3 in response to LPS andpoly(I:C) (Fig. 6), we tested this pos-sibility after immunoprecipitatingendogenousTBK1 fromLPS-stimu-lated RAW macrophages. Theseexperiments confirmed that theendogenous TBK1 was potentlyinhibited by BX795 (Fig. 6F).We also found that IL-1� (Fig.

7A) and TNF� (Fig. 7B) activatedTBK1 and IKK� in immortalizedMEFs, activation again correlatingwith the phosphorylation of Ser-172. The IL-1�-stimulated activa-tion peaked after 5–10 min anddeclined thereafter. Activation byTNF� peaked at 10min but had dis-appeared by 60 min. Similar to LPSand poly(I:C), BX795 enhanced theactivation of TBK1 and the phos-phorylation of TBK1/IKK� atSer-172 by IL-1� (Fig. 7C). BX795did not decrease the TNF�-stimu-lated activation of TBK1 or thephosphorylation of TBK1/IKK�, butin contrast to LPS, poly(I:C), andIL-1�, did not increase it (Fig. 7D).Activation of TBK1/IKK� by

TNF� but Not IL-1� Is Reduced inTAK1-deficient MEFs—TBK1 and IKK� are members of theIKK subfamily of protein kinases. To start to address the ques-tion of which protein kinase(s) activates TBK1 and IKK� incells, we therefore investigatedwhether the activation of TBK1/IKK� was affected in MEFs that express a truncated inactiveform of TAK1 (MAP3K7) (26), aMAP3K that has been impli-cated in the activation of IKK�/� by inflammatory stimuli.These experiments showed that the activation of TBK1/IKK� by IL-1� was unaffected (Fig. 8A), but a reduction inthe extent of activation after stimulation by TNF� wasobserved consistently (Fig. 8B). As expected, the phospho-rylation of p105, an established physiological substrate ofIKK�, was abolished in the TAK1-deficient MEFs (Fig. 8).

FIGURE 4. The overexpression of TBK1 and IKK� leads to autophosphorylation and transphosphorylation ofSer-172. A, wild type (WT) and two different catalytically-inactive FLAG-tagged mutants of TBK1 (TBK1-(K38A) andTBK1[D157A]) were expressed in HEK293 cells. Cell extract (40 �g protein) was then subjected to SDS-PAGE andimmunoblotted using anti-Ser(P)-172 TBK1 and anti-FLAG. B, FLAG-WT-TBK1 was transfected into HEK293 cells. 24 hlater the cells were treated without (�) or with (�) 1 �M BX795 for the times indicated. Cell extracts (40 �g protein)were then immunoblotted using anti-FLAG and anti-Ser(P)-172 TBK1 as in A. C, a vector encoding FLAG WT-TBK1was transfected into HEK293 cells, then incubated for 1 h without (�) or with (�) 1 �M BX795. TBK1 was thenimmunoprecipitated from 0.1 mg of cell extract protein with anti-FLAG (washed) and assayed for activity using theI�B� peptide substrate (mean � S.E., n � 4; *, p � 0.001), D, a vector encoding FLAG-tagged TBK1-(K38A) wasco-expressed in HEK293 cells with an empty vector (�), a vector encoding GST (�), or a vector encoding GST-WT-TBK1 (TBK1) GST-WT-IKK� (IKK�). The cells were lysed, and 40 �g of cell extract protein was immunoblotted (WB)using anti-Ser(P)-172 TBK1/IKK� or anti-FLAG as in A (upper two panels). A further 0.5 mg of cell extract protein wasincubated for 1 h at 4 °C with 0.01 ml of glutathione-Sepharose beads with continuous mixing. The beads werecollected by centrifugation and washed three times in lysis buffer, and bound proteins were released with SDS andimmunoblotted with anti-GST or anti-FLAG antibodies (lower two panels). E, wild type and catalytically-inactivemutants of FLAG-TBK1 and FLAG-IKK� were expressed in HEK293 cells, and the lysates were immunoblotted usinganti-FLAG as in A. F, WT-FLAG-IKK� was expressed in HEK293 cells. 24 h later the cells were treated with 1 �M BX795for the times indicated. Cell extracts (40 �g protein) were then immunoblotted using anti-FLAG as in A. Anti-glycer-aldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.

FIGURE 5. Endogenous IKK� protein expression is unaffected by treat-ment with BX795 and does not form a complex with TBK1. A, RAW264.7cells were treated for 1 h without (�) or with (�) 1 �M BX795 before stimula-tion for 16 h without (�) or with (�) 100 ng/ml LPS. Cell extracts were immu-noblotted with antibodies raised against IKK�, TBK1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B, endogenous TBK1 and IKK� wereimmunoprecipitated (IP) from cell extracts of RAW264.7 cells. IgG isolatedfrom a non-immunized sheep served as a negative control. Proteins presentin the immunoprecipitates were detected by immunoblotting with antibod-ies that recognize IKK�, TBK1, and TANK.

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Effect of BX795 on Activation of MAP Kinases by LPS, poly(I:C), IL-1�, or TNF�—BX795 had no effect on the LPS (Fig. 9A)-or poly(I:C)-stimulated (Fig. 9B) phosphorylation of the acti-vating Thr-Glu-Tyr motif of ERK1/ERK2. In contrast, BX795strongly suppressed the LPS (Fig. 9A)- or poly(I:C)-stimulated(Fig. 9B) phosphorylation of the activating Thr-Pro-Tyr motifon JNK1/2 and partially inhibited the phosphorylation of theactivating Thr-Gly-Tyrmotif on p38�MAP kinase. BX795 also

suppressed the phosphorylation ofJNK1/2 and p38� MAPK in MEFsstimulatedwith IL-1� orTNF� (Fig.9, C andD). As discussed below, theeffect of BX795 on the activation ofJNK1/2 and p38� MAPK does notresult from the inhibition ofTBK1/IKK�.

DISCUSSION

Small cell-permeable inhibitorsof protein kinases have proved to bevaluable reagents for identifyingnovel substrates of these enzymesand for studying their physiologicalroles, butmore compoundswith therequisite potency and specificity tobe really useful are needed. Here, weintroduce BX795 as the first phar-macological inhibitor suitable forstudying the function and regula-tion of TBK1 and IKK� in cells.TBK1 and IKK� are known to be

required for the activation of thetranscription factor IRF3 and pro-duction of Type 1 interferons (10–13). However, when overexpressedin mammalian cells, these proteinkinases also activate NF�B-depend-ent gene transcription (7–9, 18–20)(see the Introduction). In the pres-ent study we found that BX795 sup-pressed the activation of IRF3 andthe production of interferon � buthad no effect on the activation ofNF�B or NF�B-dependent genetranscription by any of the stimulithat we studied. These results,which do not support a role forTBK1 and IKK� in activating NF�Bin cells, are consistent with the phe-notype of the mouse knock-out,where the absence of TBK1 andIKK� expression also had no effecton NF�B-dependent gene tran-scription by inflammatory stimuli(10, 11). We, therefore, concludethat despite their similarity to IKK�and IKK�, the othermembers of theIKK subfamily of protein kinases,

TBK1 and IKK�, are not rate-limiting for the activation ofNF�Bby TLR3 and TLR4 agonists or by TNF� and IL-1�.

The present study is the first to show that TBK1 and IKK�can be activated by IL-1�.We found that the activation ofTBK1and IKK� by IL-1� and TNF� was rapid (5–10 min) but tran-sient. This contrasted with the slower and more sustained acti-vation of these protein kinases by poly(I:C) and LPS and mightexplain, at least in part, whyTNF� and IL-1� did not induce the

FIGURE 6. BX795 increases the phosphorylation of Ser-172 and the catalytic activity of TBK1 and IKK� inresponse to LPS and poly(I:C). A, RAW264.7 cells were treated for 1 h without (�) or with (�) 1 �M BX795before stimulation for 45 min without (�) or with (�) 100 ng/ml LPS. TBK1 was immunoprecipitated from 1 mgof cell extract protein, and its catalytic activity was measured by incubation for 30 min at 30 °C with GST-IRF3and Mg[�-32P]ATP. Reactions were terminated in SDS, proteins were resolved by SDS-PAGE, and the gel wasautoradiographed (top panel). The gel was subjected to phosphorimaging analysis, and the catalytic activity ofTBK1 was quantitated and normalized to that observed in the unstimulated cells (mean � S.E., n � 3; *, p �0.05). B, an aliquot of the cell extract in A was used to analyze the phosphorylation of TBK1 and IKK� at Ser-172as in Fig. 3C (top panel). As a loading control, cell extract (40 �g of protein) was also immunoblotted withantibodies recognizing all forms of TBK1 and IKK� (lower panel). This antibody also recognizes a nonspecificband migrating slower than TBK1 and IKK�. C, RAW264.7 cells were incubated for 1 h with the indicatedconcentrations of BX795 and then either left unstimulated (�) or stimulated (�) for 45 min with 100 ng/ml LPS.The phosphorylation of TBK1 and IKK� at Ser-172 was then examined by immunoprecipitation/immunoblot-ting as in Fig. 3C. D, bone marrow-derived macrophages were incubated for 1 h without (�) or with (�) 1 �M

BX795 followed by stimulation for 30 or 60 min with 100 ng/ml LPS. Cell extract (20 �g protein) was thenimmunoblotted with the antibodies indicated. E, the experiment was performed as in D, but the cells werestimulated for 60 min with 10 �g/ml poly(I:C). F, RAW264.7 cells were stimulated for 45 min with 100 ng/ml LPS,TBK1 was immunoprecipitated from 0.5 mg of cell extract protein, and its ability to phosphorylate GST-IRF3 (2�M) was measured at varying concentrations of BX795 using Mg[�-32P]ATP. Reactions were terminated in SDS,proteins were resolved by SDS-PAGE, and the gel was autoradiographed (top panel). The gel was subjected tophosphorimaging analysis, and the catalytic activity of TBK1 was quantitated and normalized to that measuredin the absence of inhibitor (mean � S.E., n � 4).

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phosphorylation of IRF3 or the production of interferon �(results not shown). The inability of TNF� and IL-1� to coupleto TRAF3, which is located in the endosomal compartment ofcells (33),may also account for the failure of TNF� and IL-1� toactivate the interferon pathway. The substrates of TBK1 andIKK� in the TNF� and IL-1� signaling pathways remain to beidentified, although TBK1 knock-out mice die just before birthfrom TNF-induced apoptosis of the liver (21). This suggeststhat TBK1normally plays a role in suppressingTNF-stimulatedapoptosis, at least in embryonic liver.Before the present study, others had proposed that the phos-

phorylation of TBK1 and IKK� at Ser-172 and the activation ofthese protein kinases was an autophosphorylation event (9, 15).These conclusions were based on overexpression studies, andindeed, we confirmed that the phosphorylation of Ser-172 andactivation of the overexpressed TBK1 catalytic subunit wasblocked by BX795. However, BX795 failed to suppress thephosphorylation and activation of endogenous TBK1 and IKK�in response to poly(I:C), LPS, TNF�, and IL-1�. These resultsdemonstrate that although TBK1 and IKK� can autophospho-

rylate at Ser-172 and autoactivate when they are overexpressedin cells, the activation of the endogenous protein kinases is notmediated by autophosphorylation but by a distinct upstreamactivating kinase(s). The molecular mechanisms that preventthe autophosphorylation of endogenous TBK1 and IKK� areunclear. However, endogenous TBK1 and IKK� are present asinactive and separate complexes in unstimulated cells, presum-ably due to their interactionwith other proteins, such asTANK,NAP-1, SINTBAD, and Optineurin (8, 34–36) and/or theirdephosphorylation by protein phosphatases.It will clearly be important to identify the protein kinase(s)

responsible for phosphorylating TBK1 and IKK� at Ser-172, themost likely candidates being one or more of the 22 members oftheMAP3K subfamily and the 12members of theMAP4K sub-family. LPS- and poly(I:C)-stimulated IRF3-dependent genetranscription was not affected in MEFs that do not expressTAK1 (MAP3K7) (37) and the overexpression of a dominantnegative mutant of TAK1 blocked NF�B-dependent gene tran-scription without affecting IRF3-dependent gene transcriptionin response to poly(I:C) and TLR3 agonists (37, 38). Thus,TAK1 does not seem to be required for the activation of TBK1/IKK� by LPS or poly(I:C). In the present study we showed thatthe IL-1�-mediated phosphorylation of TBK1 and IKK� at Ser-172 in MEFs that express a truncated, inactive form of TAK1instead of the wild type protein is similar to wild type MEFs.Interestingly, the TNF�-induced phosphorylation of TBK1/IKK� at Ser-172 was reduced significantly in the TAK1-defi-cient MEFs (Fig. 8), indicating that TAK1 contributes to theactivation of TBK1/IKK� by this agonist. Nevertheless, TAK1might not be a direct activator of TBK1/IKK� but is, instead,required for the synthesis or activation of another protein

FIGURE 7. Activation and phosphorylation of TBK1 and IKK� by IL-1� andTNF�. A and B, kinetics of activation of TBK1 and IKK�. MEFs were serum-starved overnight and stimulated for the indicated times using 10 ng/ml IL-1�(A) or TNF� (B). Catalytic activity was measured by immunoprecipitating TBK1and incubating the immune complexes with GST-IRF3 in the presence ofMg[�-32P]ATP. Proteins were resolved by SDS-PAGE, and phosphorylatedproteins were detected by autoradiography (top panel). Phosphorylation ofSer-172 on TBK1 was monitored by immunoprecipitating the phosphoryla-ted TBK1 and IKK� with the anti-IKK� Ser(P)-172 antibody (S051C) and immu-noblotting with an antibody recognizing all forms of TBK1 and IKK� (middlepanel). C and D, effect of BX795 on the activation of TBK1 and IKK�. MEFs wereincubated for 1 h without (�) or with (�) 1 �M BX795 before stimulation for 10min with 10 ng/ml IL-1� (C) or TNF� (D). Catalytic activity and phosphoryla-tion of TBK1 and IKK� were measured as in A and B.

FIGURE 8. TNF�, but not IL-1�, induced phosphorylation of TBK1 andIKK� is reduced in TAK1-deficient MEFs. Wild type (WT) MEFs and MEFsexpressing a truncated inactive form of TAK1 (26) were stimulated with 10ng/ml IL-1� (A) or TNF� (B) for 10 min. The phosphorylation of TBK1 and IKK�was monitored using the immunoprecipitation/immunoblotting assaydescribed in Fig. 3C. The phosphorylation of p105 as well as total TAK1 andTBK1/IKK� was detected in total protein extracts (40 �g) by immunoblottingwith the respective antibodies. TAK1� is an inactive form of TAK1, which lacks37 amino acid residues in a region critical for ATP binding. Similar results wereobtained in two independent experiments. KO, knockout.

FIGURE 9. Effect of BX795 on the activation of MAP kinases. A and B,RAW264.7 cells were treated without (�) or with (�) 1 �M BX795 and eitherleft unstimulated (�) or stimulated (�) with 100 ng/ml LPS (A) or 10 �g/mlpoly(I:C) (B). The cells were lysed, and aliquots of the lysates were subjected toSDS-PAGE and immunoblotting with antibodies that recognize the phospho-rylated forms of ERK1/2, p38� MAPK, and JNK1/2. As a loading control, cellextracts were immunoblotted with an antibody recognizing all forms of p38�MAP kinase. C and D, same as A and B, except that MEFs were deprived ofserum overnight and then incubated without (�) or with (�) 1 �M BX795 for60 min before stimulation with 10 ng/ml IL-1� (C) or TNF� (D).

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kinase that directly phosphorylates and activates TBK1/IKK�under these conditions. In conclusion, these experiments inTAK1-deficient MEFs indicate that distinct protein kinasesmay mediate the activation of TBK1/IKK� by TNF� and IL-1�.The MLK isoforms are potently inhibited by BX795 (Table

1), which may underlie the suppression of JNK1/2 and p38�MAPK activation by BX795, as discussed later. However, asBX795 does not inhibit the phosphorylation of TBK1/IKK� atSer-172, the MLKs can probably also be excluded as activatorsof TBK1/IKK�.

Our finding that the activation of endogenous TBK1 andIKK� is not an autophosphorylation event, as believed previ-ously, has more general implications, because it raises the pos-sibility that other protein kinases currently believed to be acti-vated by autophosphorylation based on overexpressionexperiments alone may also be activated by separate upstreamkinases in vivo. This issuewill need to be revisitedwhen specificpharmacological inhibitors of these protein kinases becomeavailable.Interestingly, BX795 enhanced the phosphorylation and

activation of TBK1 and IKK� by poly(I:C), LPS, and IL-1� butnot by TNF�. This finding implies that a feedback controlmechanism exists in which TBK1 and IKK� phosphorylate anupstream component(s) of this signaling pathway to limit theextent to which TBK1/IKK� can be activated, thereby avoidingthe overproduction of Type 1 interferons. Which upstreamcomponent is the target for feedback control is unknown, but ifit was the protein kinase that activates TBK1/IKK� in responseto LPS, poly(I:C), and IL-1�, this may explain why the feedbackloop does not operate in the pathway leading to the activation ofTBK1/IKK� by TNF�, which appears to require a separateand/or additional upstream activating protein kinase.MAPK cascades play important roles in regulating the pro-

duction of inflammatory mediators during bacterial and viralinfection, which led us to find that BX795 suppresses the acti-vation of p38�MAPK and JNK1/2 by LPS, poly(I:C), IL-1�, andTNF�. Two lines of evidence indicate that this effect of BX795is independent of TBK1/IKK�. First, the activation of theseMAPKs by poly(I:C) is not impaired inMEFs from TBK1/IKK�double knock-out mice (10). Second, novel chemical entitieshave recently been derived from BX795 by our collaborators atMRC Technology, which retain their ability to inhibit TBK1/IKK� and to suppress the phosphorylation of IRF3 and produc-tion of interferon � but no longer inhibit the activation of p38�MAPK or JNK1/2 in cells.4 The immediate upstream activatorsof JNK1/2 are MKK4 and MKK7 and for p38 MAP kinases areMKK3 and MKK6, but these four MKKs were unaffected byBX795 in vitro at concentrations (1 �M) that suppress the acti-vation of JNK1/2 and p38� MAPK in cells. This suggests thatthe target of BX795 in the p38�MAPK and JNK1/2 pathways isupstream of the MKKs.LPS, IL-1�, and TNF� are unable to activate the IKK�/�

complex, JNK1/2, and p38 MAPKs in TAK1-deficient MEFs(26, 37), and these findings have led to a widespread acceptancethat TAK1 is the direct activator of IKK�/� as well as theMKKs

that activate JNK1/2 and p38MAPK, in response to these stim-uli. However, in the present study, we found that BX795 sup-pressed the activation of JNK1/2 but had no effect on the acti-vation of the IKK�/� complex by inflammatory stimuli. Theseobservations are inconsistent with the notion that the sameMAP3K activates both signaling pathways but are consistentwith the work of Zhong and Kyriakis (39, 40). These investiga-tors reported that LPS and some other inflammatory stimuliactivate JNK1/2 and p38 MAPK by stabilizing MAP4K2, alsotermed germinal centre kinase (39). This led to a rapid rise inthe expression of germinal centre kinase, enabling it to activateMLK2 (MAP3K10) and MLK3 (MAP3K11), which then acti-vated the MKKs that switch on JNK1/2 and p38 MAPK. Incontrast, the germinal centre kinase-MLK2/3 pathway does notactivate the IKK�/� complex (40). These findings led us to dis-cover that BX795 is a potent inhibitor of MLK isoforms in vitro(Table 1). Moreover, the novel compounds that have recentlybeen derived from BX795 byMRCTechnology and do not sup-press the activation of JNK1/2 and p38� MAPK in cells are farless potent inhibitors of MLK3 in vitro than BX795.4 Theseresults suggest that inhibition of one or more MLK isoformsmay underlie the inhibition of JNK1/2 and p38�MAPKby LPS,poly(I:C), IL-1�, and TNF�. In contrast, BX795was found to bea much weaker inhibitor of TAK1 in vitro (Table 1), whichcould explain why BX795 does not suppress the activation ofIKK�/� complex. Why inflammatory stimuli cannot activateJNK1/2 and p38� MAPK in TAK1-deficient cells is unknown,although one possible explanation is that TAK1 controls the4 K. Clark, L. Plater, and P. Cohen, unpublished results.

FIGURE 10. Model for the activation and feedback control of TBK1 and IKK�by proinflammatory stimuli. The binding of IL-1�, LPS, and poly(I:C) to theirrespective receptors induces the activation of an unidentified protein kinase(PKX). PKX subsequently phosphorylates TBK1 and IKK� at Ser-172, thereby trig-gering their activation. TBK1 and IKK� can then phosphorylate substrates such asthe transcription factor IRF3. As shown in this study, TBK1 and IKK� also exert anegative feedback control on their activation by IL-1�, LPS, and poly(I:C) presum-ably by phosphorylation and inhibition of an upstream component of the path-way and/or by activating a Ser-172 protein phosphatase (not illustrated). TheTNF�-stimulated activation of TBK1 and IKK� appears to require a separate oradditional protein kinase that is at least partially dependent on TAK1 activity, andthis arm of the pathway is not subject to feedback control.

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expression of one or more germinal centre kinase and/or MLKisoforms.In summary, we have used the pharmacological inhibitor

BX795 to study the function and regulation of TBK1 and IKK�.Most importantly, our results have demonstrated that the acti-vation of endogenous TBK1 and IKK�, which correlates withthe phosphorylation of Ser-172, is not an autophosphorylationevent. Our current paradigm for the activation of TBK1 andIKK� in response to proinflammatory stimuli is summarized inFig. 10.Upon stimulation of cellswith LPS, poly(I:C), IL-1�, andTNF�, protein kinases are activated that phosphorylate Ser-172in the activation loop of TBK1 and IKK�. LPS, poly(I:C), andIL-1� control a negative feedback loop that prevents the hyper-activation of TBK1 and IKK� by phosphorylating one or moreupstream components of these pathways. Alternatively, or inaddition, the feedback control loop might involve the TBK1/IKK�-mediated activation of a protein phosphatase(s) thatdephosphorylates Ser-172. This feedback control loop does notoperate in the TNF� signaling pathway in MEFs, which seemsto employ a distinct upstream protein kinase for the activationof TBK1 and IKK� that is dependent on TAK1 activity.

Acknowledgments—We thankDr. Katherine Fitzgerald for generouslyproviding IRF3 andNF�B reporter constructs and the 293-TLR3 cells.We also thank Professor Shizuo Akira for kindly providing immortal-izedMEFs expressing a truncated, inactive form of TAK1 and controlMEFs, Dr. Alan Prescott for assistance with microscopy, Dr. NataliaShpiro for synthesizing BX795, and the Protein andAntibody Produc-tion Teams of the Division of Signal Transduction Therapy at theUniversity of Dundee (coordinated by HilaryMcLauchlan and JamesHastie) for proteins and antibodies.

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Activation of TBK1 and IKK� by Upstream Kinase

14146 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 21 • MAY 22, 2009

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Kristopher Clark, Lorna Plater, Mark Peggie and Philip CohenKINASE MEDIATES SER-172 PHOSPHORYLATION AND ACTIVATION

B Kinase ?: A DISTINCT UPSTREAMκPhysiological Roles of TBK1 and IUse of the Pharmacological Inhibitor BX795 to Study the Regulation and

doi: 10.1074/jbc.M109.000414 originally published online March 22, 20092009, 284:14136-14146.J. Biol. Chem. 

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